Effect Of Nitric Oxide Production Research Articles

Characterization of low-molecular-weight fucoidan prepared by photocatalysis degradation and its regulation effect on metabolomics of LPS-stimulated macrophages

Fucoidan (ANP) from Ascophyllum nodosum demonstrates significant anti-inflammatory activity. Because lower-molecular-weight polysaccharides usually process even better bioactivities, in the present study, the photocatalytic degradation method with TiO2 and H2O2 was used to prepare low-molecular-weight derivatives (D-ANP-1.5 and D-ANP-3.5) with molecular weight of 5.0 kDa and 2.3 kDa, respectively. The characteristics of D-ANP-1.5 and D-ANP-3.5 were identified by chemical composition analysis, high-performance gel permeation chromatography, high-performance liquid chromatography photo-diode array mass, and Fourier transform-infrared spectrum, and compared with that of original fucoidan. The sulfate group retention rates of D-ANP-1.5 and D-ANP-3.5 were over 90.0% and the fucose and total sugar contents increased. However, the uronic acid and protein contents were decreased and the functional groups were not changed during degradation. Subsequently, 7 oligosaccharides in D-ANP-3.5 were identified by high-performance liquid chromatography-electrospray ionization detector mass and 6 of them were sulfated. Moreover, D-ANP-3.5 (2.3 kDa) supplement showed stronger inhibition effect of nitric oxide production than D-ANP-1.5 and undegraded ANP in LPS-stimulated RAW 264.7 cells. In addition, metabolic profiles results indicated that arginine biosynthesis, phenylalanine, tyrosine, and tryptophan biosynthesis, the TCA cycle, purine metabolism, and glutathione metabolism, along with influencing metabolic biomarkers such as betaine, arginine, NAD+, ATP, and pyroglutamic acid, were involved in the effects of D-ANP-3.5. The superior regulation effect on metabolomics in LPS-stimulated RAW 264.7 cells of D-ANP-3.5 could be attributed to its relatively low molecular weight and high fucose content. The present study advance understanding of the mechanism for the anti-inflammation of D-ANP-3.5.

Labdane diterpenoids from Lagopsis supina and their anti-neuroinflammatory activity

In this study, ten labdane-type diterpenoids 1–10 were isolated from a methanol extract of the whole plant Lagopsis supina, including three undescribed compounds 1–3. Their structures were determined by spectroscopic data analyses such as HR-ESI-MS, 1D, and 2D NMR, as well as comparison with literature data. At the same time, the absolute configuration of five compounds 2–5 and 10 was confirmed for the first time by the single crystal X-ray diffraction method. All the compounds were isolated from L. supina for the first time. The CCK-8 assay showed that all compounds had no significant damage to BV-2 microglial cells, and then screened their inhibitory effects of nitric oxide production stimulated by lipopolysaccharide in BV-2 microglial cells. The pharmacological results showed that compound 4 greatly inhibited LPS-stimulated NO release at the concentration of 10 μM, indicating that it has potential anti-neuroinflammatory activity.

Acellular hemoglobin impairs calcium release and contractility in cardiomyocytes from healthy animals

Hemoglobin (Hb)-based oxygen (O 2 ) carriers (HBOC) have been proposed as an alternative for blood transfusion. Phase III clinical trials of previous HBOC generations presented issues, such as vasoconstriction and myocardial infarction, that were not detected in pre-clinical trials. These problems have been attributed to extravasation of the low molecular weight (LMW) Hb species, nitric oxide (NO) scavenging and O 2 oversupply to the vasculature. However, the mechanisms responsible for the cardiac toxicity of HBOCs remain unclear. To explore the effects of acellular (acell) Hb and HBOC in cardiac tissues, we exposed cardiomyocytes (CMCs) to highly purified stroma-free acell bovine Hb (bHb) or human Hb (hHb). CMCs were loaded with Ca2+ fluorescent indicator (fluo-3) and put on laminin-coated coverslips. Cells were superfused with standard Tyrode solution with and without acell Hb. CMCs were stimulated with current pulses given at 1 Hz, and free cytosolic Ca2+ concentration was measured. To eliminate the effects of NO scavenging on CMCs, L-Name was added to the superfused with bHb and hHb. To study the effect of acell Hb MW on CMCs, LMW polymerized hHb (PolyhHb) was used to challenge CMCs. To explore the effect of Hb oxidation on CMCs, LMW MetHb was used to challenge the CMCs and compared to LMW PolyhMetHb. To explore the effects of antioxidants and heme toxicity, the studies with hHb and MethHb were repeated in the presence of the antioxidant N-acetylcysteine (NAC) or purified human Haptoglobin (Hp). Mitochondrial reactive oxygen species (ROS) production was measured using the fluorescent indicator MitoSOX Red after removing the Hb by washing the cells. After ROS determination, the maximal ROS production by CMCs was measured by challenging the cells with a high concentration of H 2 O 2 . Acell Hb increased the Ca 2+ transient decay time constant (tau) relative to control. Removing the effects of NO production with L-Name, increased the tau of the Ca 2+ transient in control and acell Hb challenge cells. Challenging CMCs with PolyhHb reduced the effects observed with acell Hb in Ca 2+ transients. MetHb appears to be worse than reduced Hb in terms of the changes induced to CMCs Ca 2+ transients. The antioxidant NAC mitigated the effects of acell Hb and MetHb but did not eliminate the changes on increased Ca2+ transients. The Hb scavenger, Hp, mitigated the effects of acell hHb and MethHb on Ca 2+ transients. The MitoSOX Red fluorescence was higher for CMCs exposed to acell Hb and MetHb compared to the control. PolyhHb and PolyMetHb challenge decreased the MitoSOX compared to acell hHb and MethHb. Cells with higher levels of ROS showed lower changes in fluorescence when exposed to H 2 O 2 . Here, we show that the toxicity of acell Hb is impart determined by facilitated diffusion, NO scavenging, heme release, and ROS, potentially affecting the energy supply and metabolism of CMCs. This study was supported by National Institutes of Health grants R01HL162120, R01HL159862 and the Department of Defense under grants W81XWH1810059. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Effects of Nitric Oxide Production and Lymphedema Treatment on A Pregnant Female with A History of Recurrent Abortion: A Case Report

The Effects of Nitric Oxide Production and Lymphedema Treatment on A Pregnant Female with A History of Recurrent Abortion: A Case Report

Separation and anti-inflammatory evaluation of phytochemical constituents from Pleurospermum candollei (Apiaceae) by high-speed countercurrent chromatography with continuous sample load.

Pleurospermum (Apiaceae) species possess a wide range of biological properties viz. analgesic, anti-inflammatory, antimalarial, and so on. Pleurospermum candollei (DC.) Benth. Ex C. B. Clark. is reported to cure diarrhea, gastric, respiratory, stomach, abdominal, joint, and back pain problems. In addition, it is also used for both male and female infertility. The present study deals with an efficient technique using high-speed countercurrent chromatography for separation of chemical components from the methanol extract of P. candollei. Notably, nine main compounds namely luteolin 7-O-glucoside (1), oxypeucedanin hydrate (2), pabulenol (3), bergapten (4), heptadecanoic acid (5), (E)-isoelemicin (6), trans-asarone (7), α-linolenic acid (8), and isoimperatorin (9) were very efficiently separated and isolated in pure form. Multiple injections were applied followed by two off-line recycling high-speed countercurrent chromatography. The inhibitory effect of nitric oxide production of all compounds was tested in the presence of 200ng/mL lipopolysaccharide in RAW264.7 mice macrophage cells. The results demonstrated that compounds 7 and 8 effectively inhibited nitric oxide production, with IC50 values of 28.44 and 53.18μM, respectively. This study thus validates the traditional claim of using P. candollei. Taken together, these findings will be useful in future research to find a potential candidate with anti-inflammatory properties.

Chemical constituents from Curcuma longa L. and their inhibitory effects of nitric oxide production

A new bisabolane-type sesquiterpenoid, turmerone Q (1), along with six known compounds (2–7), were isolated from the rhizomes of Curcuma longa L. The structural elucidation of the new compound was conducted using 1H NMR, 13C NMR, HSQC, HMBC and NOESY spectroscopic analyses. The absolute configuration of 1 was elucidated by comparison of the experimental and calculated ECD spectra. The anti-inflammatory effects of 1–7 were evaluated through lipopolysaccharide-induced nitric oxide (NO) production in RAW 264.7 macrophages assays, and compounds 6 and 7 showed potent inhibitory activity against NO production.

Effects of Fucoidan on Nitric Oxide Production and Activator Protein-1 Activation in Lipopolysaccharide-Stimulated Porcine Peripheral Blood Mononuclear Cells

Fucoidan which is sulfated polysaccharide extracted from brown seaweed has a wide variety of internal biological activities. The objectives of this study were to examine the effect of fucoidan on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated porcine peripheral blood mononuclear cells (PBMCs) and to investigate whether this effect is involved in the expression of inducible nitric oxide synthase (iNOS) and the activation of activator portein-1 (AP-1). The levels of NO production and AP-1 activity in the culture supernatants from porcine PBMCs were measured by the enzyme-linked immunosorbent assay and the levels of iNOS and AP-1 mRNA were determined by real time polymerase chain reaction. Fucoidan in LPS-naive PBMCs has no effects on the production of NO and activity of AP-1. Expressions of iNOS and AP-1 mRNA in LPS-naive PBMCs were also not affected by treatment of fucoidan. However, NO production, AP-1 activity and expressions of iNOS and AP-1 mRNA were dramatically increased in PBMCs stimulated with LPS. Enhancing effects of NO production and AP-1 activity in PBMCs induced by LPS were reduced by addition of fucoidan. Fucoidan also inhibited an increase in expressions of iNOS and AP-1 mRNA in LPS-stimulated PBMCs. These results suggested that fucoidan exerts anti-inflammatory effect by down-regulating production of NO via suppressing expression of iNOS and activity of AP-1 in LPS-stimulated porcine PBMCs.

Abstract 4712: Targeting the arginine-nitric oxide pathway to arrest development and progression of invasive bladder cancer

Abstract Background: In United States there will be an estimated 74,690 new diagnoses and 15,580 deaths in 2014 due to bladder cancer. Lack of reliable markers of early disease and limited chemotherapy options have resulted in poor patient outcomes. A better approach is to treat patients with early intervention using targeted therapy. Arginine metabolic pathway has been identified to be deregulated in bladder cancer. Arginine is either derived from the microenvironment or synthesized by cells containing arginosuccinate synthase I (ASS1). Arginine undergoes subsequent conversion to nitric oxide (NO), which which can activate numerous intracellular signaling mediators, including guanylate cyclase (GC). We evaluated dual targeting of this metabolic pathway by arginine deprivation (ADI-PEG 20, Polaris Pharmaceuticals, San Diego, CA) and NO chelation (Cobinamide) as a therapy for bladder cancer. Experimental Design: Expression of arginine-NO pathway components in FFPE tissue sections was assessed by IHC and correlated with histopathology, outcomes and progression to identify pathway activation during early and late disease and stratified based on moderate to strong expression (2+, 3+) compared to absent to weak expression (0, 1+). In addition, a panel of bladder cell lines was screened for arginine-NO pathway components by immunoblotting and representative ASS1 negative and positive cells were used to evaluate the effects of arginine deprivation and/or NO chelation in vitro on colony formation and cell viability by clonogenic and MTT assays. Effects of NO production on bladder cancer survival and motility was assessed utilizing siRNA gene silencing and small molecule inhibitors. Effects of arginine deprivation by ADI-PEG 20 treatment in vivo was assessed by a mouse xenograft model. Results: Analysis of human tumors showed that ASS1 loss and increase in NOS occurred early in the course of bladder neoplastic progression. We identified loss of the ASS1 enzyme in bladder cancer cells and compensatory dependence on extracellular arginine, which can be successfully targeted using arginine removal to induce bladder cancer cell death, both in vitro and in vivo. We also identified increased nitric oxide synthase (NOS; the enzyme that catalyzes NO production) and GC activation in invasive bladder cancer cell lines. We characterized the effects of NO production on bladder cancer cell survival, growth and motility. Disruption of the NO pathway through gene silencing and small molecule inhibitors resulted in decreased cell survival, migration and invasion. Conclusions: Thus, dual targeting of this metabolic pathway using arginine chelation by the arginine degrading enzyme ADI-PEG20 and NO scavenging by a novel compound Cobinamide can be a potential mechanism to prevent development and/or progression of early bladder neoplasia. Citation Format: Divya Sahu, Sounak Gupta, Andrew M. Hau, Paul Elson, John Bomalaski, Gerry R. Boss, Donna E. Hansel. Targeting the arginine-nitric oxide pathway to arrest development and progression of invasive bladder cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4712. doi:10.1158/1538-7445.AM2015-4712

Anti-Inflammatory Effect of Neoechinulin A from the Marine Fungus Eurotium sp. SF-5989 through the Suppression of NF-кB and p38 MAPK Pathways in Lipopolysaccharide-Stimulated RAW264.7 Macrophages

In the course of a bioassay-guided study of metabolites from the marine fungus Eurotium sp. SF-5989, two diketopiperazine type indole alkaloids, neoechinulins A and B, were isolated. In this study, we investigated the anti-inflammatory effects of neoechinulins A (1) and B (2) on lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Neoechinulin A (1) markedly suppressed the production of nitric oxide (NO) and prostaglandin E2 (PGE2) and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in a dose dependent manner ranging from 12.5 µM to 100 µM without affecting the cell viability. On the other hand, neoechinulin B (2) affected the cell viability at 25 µM although the compound displayed similar inhibitory effect of NO production to neoechinulin A (1) at lower doses. Furthermore, neoechinulin A (1) decreased the secretion of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). We also confirmed that neoechinulin A (1) blocked the activation of nuclear factor-kappaB (NF-κB) in LPS-stimulated RAW264.7 macrophages by inhibiting the phosphorylation and degradation of inhibitor kappa B (IκB)-α. Moreover, neoechinulin A (1) decreased p38 mitogen-activated protein kinase (MAPK) phosphorylation. Therefore, these data showed that the anti-inflammatory effects of neoechinulin A (1) in LPS-stimulated RAW264.7 macrophages were due to the inhibition of the NF-κB and p38 MAPK pathways, suggesting that neoechinulin A (1) might be a potential therapeutic agent for the treatment of various inflammatory diseases.

HBOC Vasoactivity: Interplay Between Nitric Oxide Scavenging and Capacity to Generate Bioactive Nitric Oxide Species

Despite many advances in blood substitute research, the development of materials that are effective in maintaining blood volume and oxygen delivery remains a priority for emergency care and trauma. Clinical trials on hemoglobin (Hb)-based oxygen carriers (HBOCs) have not provided information on the mechanism of toxicity, although all commercial formulations have safety concerns. Specifically, it is important to reconcile the different hypotheses of Hb toxicity, such as nitric oxide (NO) depletion and oxidative reactions, to provide a coherent molecular basis for designing a safe HBOC. HBOCs with different sizes often exhibit differences in the degree of HBOC-induced vasoactivity. This has been attributed to differences in the degree of NO scavenging and in the extent of Hb extravasation. Additionally, it is appears that Hb can undergo reactions that compensate for NO scavenging by generating bioactive forms of NO. Engineering modifications to enhance bioactive NO production can result in diminished oxygen delivery by virtue of increased oxygen affinity. This strategy can prevent the HBOC from fulfilling the intended goal on preserving oxygenation; however, the NO production effects will increase perfusion and oxygen transport. Hb modifications influence NO scavenging and the capacity of certain HBOCs to compensate for NO scavenging through nitrite-mediated reactions that generate bioactive NO. Based on the current understanding of these NO-related factors, possible synthetic strategies are presented that address how HBOC formulations can be prepared that: (i) effectively deliver oxygen, (ii) maintain tissue perfusion, and (iii) limit/reverse underlying inflammation within the vasculature.

Apoptotic mechanisms are involved in the death of Strongyloides venezuelensis after triggering of nitric oxide

Despite progress in understanding the role of nitric oxide (NO) in the pathogenesis of helminth infections, the role in strongyloidosis is unknown. Firstly, we studied the production of NO in mice infected with Strongyloides venezuelensis as well as in macrophage cultures stimulated with parasite antigens. Somatic larvae 3 (L3) and excretory-secretory female antigens stimulate specific NO production measured by Griess reaction and expression of inducible NO synthase by RT-PCR and quantitative PCR. Moreover, mice infected with S.venezuelensis produce NO in migration stages. Secondly, we analysed the effect of NO production on L3 and females of S.venezuelensis using NO donors such as diethylenetriamine and 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene. Parasites died after NO donor treatment in a dose-dependent manner. Finally, apoptotic mechanisms are involved in the death of S.venezuelensis larvae.

The detrimental effect of nitric oxide on tissue is associated with inflammatory events in the vascular endothelium and neutrophils in mice with dextran sodium sulfate-induced colitis

Nitric oxide (NO) is thought to be a key molecule in the progression of ulcerative colitis and experimental colitis induced by dextran sodium sulfate (DSS). However, the detrimental effect of DSS-induced NO production on the colonic mucosa is incompletely understood. Increases in the expression of adhesion molecules in the vascular endothelium and activated neutrophils (thereby releasing injurious molecules such as reactive oxygen species) are reportedly associated with the pathogenesis of DSS-induced colitis. We investigated if the detrimental effect of NO production on the colonic mucosa was attributable to the activation of neutrophil infiltration by NO in mice with DSS-induced colitis. NO2−/NO3− content in the middle and distal colon was increased on days 5 and 7, but alterations in the proximal colon were not observed. Myeloperoxidase (MPO) activity and expression of P-selectin and intercellular adhesion molecule-1 (ICAM-1) were significantly increased in the entire colon, whereas TNF-α levels were significantly increased only in the middle and distal colon on day 7. The pathology of colitis and increases in colonic MPO activity, P-selectin, ICAM-1, and TNF-α levels were suppressed by the inducible NO synthase (iNOS)-specific inhibitor aminoguanidine and NO scavenger c-PTIO, whereas all but TNF-α levels were increased by the non-specific NOS inhibitor L-NAME. These findings suggest that iNOS-derived NO increases TNF-α levels in the middle and distal colon and increased TNF-α levels induce expression of P-selectin and ICAM-1, thereby promoting the infiltration of activated neutrophils, which leads to damage to colonic tissue.

Characterization of fructan from Chikuyo-Sekko-To, a Kampo prescription, and its antiherpetic activity in vitro and in vivo

A fructan (CSH1-1, Mw = 4.0 × 10 3) was isolated as an antiviral principle by combination of column chromatographies from Chikuyo-Sekko-To (CST), which is a traditional Japanese herbal (Kampo) medicine for infectious diseases. Chemical and spectroscopic analyses revealed that CSH1-1 was a highly branched fructan consisted of terminal (19.0%), 2,1- (61.9%), 2,6- (4.9%) and 1,2,6-linked β- d-Fru f residues (12.3%) with 1,6-linked β- d-Glc p residues (1.9%). CSH1-1 showed anti-herpes simplex virus type 2 (HSV-2) effects in vitro and in vivo. The polysaccharide had stimulating effects of nitric oxide production and induction of several cytokine mRNA expression including IL-1β, IL-6, IL-10 and TNF-α on RAW264.7 cells. From these results, a fructan from CST was suggested to be a candidate as an anti-HSV-2 agent.

Effects of Vitex rotundifolia on radical scavenging and nitric oxide production

Vitex rotundifolia (V. rotundifolia) has been used for treating headache, dizziness, toothache and removal of fever as a traditional medicine in Korea. In the present study, we examined the antioxidant and anti-inflammatory activities of 85% methanol extract of V. rotundifolia. In various radical scavenging assays, V. rotundifolia exhibited strong scavenging effect on 1, 1-diphenyl-2-picrylhydrazyl free radical, superoxide radical, nitric oxide. To elucidate the anti-inflammatory properties of V. rotundifolia, we investigated the inhibition effects of nitric oxide production in IFN-gamma and LPS-stimulated mouse peritoneal macrophages. V. rotundifolia suppressed nitric oxide production, iNOS and COX-2 expression dose-dependently through suppression of NF-<TEX>$\hat{e}B$</TEX> activation without notable cytotoxicity. These findings mean that V. rotundifolia may be beneficial in oxidative stress-mediated inflammatory disorders.

Protective effect of Neorhodomela aculeata methanolic extract through the suppressive action on NF-κB and STAT pathway in IL-1β and IFN-γ induced β-cell damage

The protective effects of Neorhodomela aculeata methanolic extract (N. aculeata) on IL-1β and IFN-γ induced β-cell cytotoxicity were examined in a rat insulinoma (RIN) cell line and islets. In the cytokine-treated RIN cells, nitric oxide (NO) production was reduced about 94% at 50 μg/ml of N. aculeata and its suppressive effect was ten times greater as compared to other effective oriental herbs, such as C. cinnamoni and A. capillaries. The inhibitory effects of NO production occurred through the suppression of iNOS (inducible nitric oxide synthase) gene expression which has been confirmed with the changes of iNOS mRNA and protein levels. N. aculeata attenuated the cytokine-induced increase in nuclear factor-kappa B (NF-κB) binding activity, p50 and p65 subunit levels in their nuclei, and at inhibitory kappa B alpha degradation in the cytosol. N. aculeata also attenuated the levels of phosphorylated signal transducer and activator of transcription (STAT)-1 and -3 in whole RIN cells as well as the nuclear translocation of the STAT proteins. Therefore, the cytoprotective effects of N. aculeata were possibly mediated through the suppression of NF-κB and STAT pathways. These findings suggest the beneficial effects of N. aculeata when used for the prevention or attenuation of inflammatory and Type 1 diabetogenic processes.

Evaluation of anti-inflammatory effect of fucoxanthin isolated from brown algae in lipopolysaccharide-stimulated RAW 264.7 macrophages

In this study, potential anti-inflammatory effect of fucoxanthin isolated from brown algae was assessed via inhibitory effect of nitric oxide (NO) production in lipopolysaccharide (LPS) induced RAW 264.7 macrophage cells. The Myagropsis myagroides was selected for further experiments due to its profound NO inhibitory effect, and was partitioned with different organic solvents. Highest NO inhibitory effect was detected in the chloroform fraction, and the active compound was identified as fucoxanthin, a kind of carotenoid available in brown algae evidenced high correlation with the inhibitory effect of NO production ( r 2 = 0.9511). Though, fucoxanthin significantly inhibited the NO production, it slightly reduced the prostaglandin E 2 (PGE 2) production. The inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) protein expressions were inhibited by fucoxanthin. Further, RT-PCR analysis indicated that the iNOS and COX-2 mRNA expressions were suppressed by fucoxanthin. Moreover, the release of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), and the mRNA expression levels of those cytokines were reduced by the addition of fucoxanthin in a dose-dependent manner. Hence, these results suggest that the use of fucoxanthin may be a useful therapeutic approach for the various inflammatory diseases.

Protein nitration is associated with increased proteolysis in skeletal muscle of bile duct ligation–induced cirrhotic rats

Cirrhosis is characterized by skeletal muscle wasting. In this study, the effects of nitric oxide production on skeletal muscle protein nitration and degradation in cirrhosis were investigated. Cirrhosis was induced by bile duct ligation (BDL) in Sprague-Dawley rats for 4 weeks. The BDL-induced cirrhotic rats and sham-operated rats were then injected daily with either saline or N G - l-nitro-arginine methyl ester ( l-NAME) for 7 days from week 4 to week 5, after which nitrite/nitrate, glutathione reduction, as well as protein nitration, ubiquitination, and degradation were assessed in skeletal muscle. Elevated muscular nitrite/nitrate concentrations, protein nitration, total ubiquitin conjugates, and degradation fragments of myosin heavy chain as well as diminished glutathione reduction levels were observed in BDL-induced cirrhotic rats as compared with controls. Administration of l-NAME for 1 week led to reduction of nitrite/nitrate levels; protein nitration was also decreased in the skeletal muscle. In addition, ubiquitination of muscular proteins and degradation of myosin heavy chain were significantly diminished after treatment of l-NAME. In conclusion, nitrosative stress occurred in the skeletal muscle of BDL-induced cirrhotic rats and may lead to increased proteolysis of muscle-specific structural proteins.

Nitric oxide synthase mediates regulation of cell polarity and movement during Drosophila melanogaster morphogenesis

The effect of expression of the enzyme producing nitric oxide on Drosophila melanogaster morphogenesis was studied using ectopic expression of a truncated splice form of the enzyme that has a dominant negative effect on the full-length enzyme form. The suppressed enzyme expression was shown to affect the cell polarity and movement as well as the structural pattern of organs during Drosophila development. The effect of nitric oxide production on the intercellular distribution of significant factors of cell polarity, protein products of the Strabismus and Prospero genes, has been revealed.

Preventing Extracellular Diffusion of Trigeminal Nitric Oxide Enhances Formalin-induced Orofacial Pain

Nitric oxide (NO), a diffusible gas, is produced in the central nervous system, including the spinal cord dorsal horn and the trigeminal nucleus, the first central areas processing nociceptive information from periphery. In the spinal cord, it has been demonstrated that NO acts as pronociceptive or antinociceptive mediators, apparently in a concentration-dependent manner. However, the central role of NO in the trigeminal nucleus remains uncertain in support of processing the orofacial nociception. Thus, we here investigated the central role of NO in formalin (3%)-induced orofacial pain in rats by administering membrane-permeable or -impermeable inhibitors, relating to the NO signaling pathways, into intracisternal space. The intracisternal pretreatments with the NO synthase inhibitor L-NAME, the NO-sensitive guanylate cyclase inhibitor ODQ, and the protein kinase C inhibitor GF109203X, all of which are permeable to the cell membrane, significantly reduced the formalin-induced pain, whereas the membrane-impermeable NO scavenger PTIO significantly enhanced it, compared to vehicle controls. These data suggest that an overall effect of NO production in the trigeminal nucleus is pronociceptive, but NO extracellularly diffused out of its producing neurons would have an antinociceptive action.

Polyaspartoyl·L-arginine enhances prostacyclin synthesis in rat aortic endothelial cells

Nitric oxide (NO) and prostacyclin (PGI 2) are two of the most important vasodilators produced by endothelial cells, the regulation of NO on PGI 2 production has not been fully clear yet. Polyaspartoyl·L-arginine (PDR) is an L-arginine residue-rich compound with inhibitory effects of platelet aggregation and thrombosis. This study investigated its effects on NO production in rat aortic endothelial cells (RAECs) and observed the influence of NO on PGI 2 level in RAECs. NO concentration in the medium of RAECs was assessed with fluorometric method; 6-keto-PGF 1α, the stable metabolite of PGI 2, in the medium of RAECs was measured with radioimmunoassay kits; Protein level of PGI 2 synthase in RAECs was determined by Western blot analysis. PDR (17.0 ~ 170 µg/ml, equal to 0.5 µM – 5 µM) enhanced NO level in culture medium of RAEC with concentration-dependent manner ( P < 0.01); L-arginine (170 µg/ml, equal to 1000 µM) and 1.70 µg/ml (0.05 µM) of PDR slightly increased NO level ( P > 0.05). Interestingly PDR (1.70 – 500 µg/ml), L-arginine (17.0 – 170 µg/ml) significantly elevated PGI 2 levels in medium of RAECs ( P < 0.01), NO synthase inhibitor, N G-nitro L-arginine methyl ester (L-NAME), markedly inhibited the elevated PGI 2 levels by PDR and L-arginine. NO donor, sodium nitroprusside(SNP)(1 – 500 µM), showed the most powerful effects of increasing PGI 2 level in RAECs, which was not influenced by L-NAME. Cyclooxigenase(COX) inhibitor, indomethacin, significantly reduced elevated PGI 2 level by both PDR and SNP in RAEC medium. PDR (170 µg/ml) increased the expression of PGI 2 synthase, L-NAME partly inhibited this effect. In conclusion, PDR enhances PGI 2 synthesis in RAEC, which is attributed to its effect of NO production; the stimulating effect of PDR on PGI 2 synthesis may be mediated via COX and PGI 2 synthase.